Laser-processed substrate for molecular diagnostics

ABSTRACT

Apparatus for use in performing a diagnostic assay of an analyte, the apparatus comprising a base that has been structured using laser processing so as to provide at least one patterned surface by melting and resolidification of the base, wherein the patterned surface is characterized by structures ranging in scale from 10 to 2000 nanometers and further wherein the pattern is stochastic in all three spatial dimensions; and a metal applied to the at least one patterned surface so as to provide at least one metalized patterned surface.

REFERENCE TO PENDING PRIOR PATENT APPLICATIONS

This patent application:

(i) is a continuation-in-part of prior U.S. patent application Ser. No.11/452,729, filed Jun. 14, 2006 now U.S. Pat. No. 7,586,601 by Steven M.Ebstein for APPLICATIONS OF LASER-PROCESSED SUBSTRATE FOR MOLECULARDIAGNOSTICS, which patent application in turn claims benefit of pendingprior U.S. Provisional Patent Application Ser. No. 60/690,385, filedJun. 14, 2005 by Steven M. Ebstein for APPLICATIONS OF LASER-PROCESSEDSUBSTRATE FOR MOLECULAR DIAGNOSTICS; and

(ii) claims benefit of pending prior U.S. Provisional Patent ApplicationSer. No. 61/125,453, filed Apr. 25, 2008 by Steven M. Ebstein forAPPLICATIONS OF LASER-PROCESSED SUBSTRATE FOR MOLECULAR DIAGNOSTICS.

The three above-identified patent applications are hereby incorporatedherein by reference.

FIELD OF THE INVENTION

The present invention pertains to an apparatus and a method forquantitatively detecting and distinguishing between compounds,especially biomolecules, with high sensitivity and selectivity using alaser-processed substrate and an optical source and detector.

BACKGROUND OF THE INVENTION

Molecular diagnostics are an increasingly important part ofbiotechnology. The ability to detect small quantities of genomic,proteomic, and other biological materials enables sensitive clinicaltests to be performed, as well as enabling laboratory research thataffects drug development and functional biology. Molecular sensingmodalities include radioactivity, mass spectroscopy, and electrical andoptical techniques. The worldwide market for molecular biomarkers,diagnostics, and related services exceeded $6 billion in 2003.

While current technology is quite useful, it is not as sensitive or asspecific as is desired, and there is room for improvement in the speedand economics of the diagnostics. The utility of many current genomicassays is limited by the need to replicate sample material usingtechniques like Polymerase Chain Reaction (PCR), which can addinaccuracies, take time to process, and may add cost through royaltypayments. Even with large material samples, the results of gene chipreaders have significant statistical variation. Consequently, there isgreat interest in techniques that improve the ability to unambiguouslydetect specific molecules in very small quantities. Nanotechnology hasbeen identified as one approach that can potentially improve thesensitivity of molecular diagnostics.

My new approach, which will hereinafter be discussed, is to use a novelfamily of nanostructured substrates to develop a sensitive opticaltechnique for molecular diagnostics that uses Surface Enhanced RamanScattering (SERS) or other optical detection techniques. Ramanspectroscopy is a non-invasive technique that requires little materialpreparation and can provide an essentially unique signature forbiological and many other molecules. Nanometer-scaled conductingstructures, through coupling to surface plasmon modes, can greatlyenhance the Raman signal so that the diagnostic can be performed in apractical setting with minute amounts of material.

I will hereinafter discuss the use of a novel nanostructured substratethat satisfies all of the requirements for successful application ofSERS to molecular diagnostics. Namely, the novel nanostructuredsubstrate is inexpensive to produce, it can be precisely tailored formaximally enhancing the Raman signal, and it can easily bemicropatterned for use as an array. As I detail below, the novelsubstrate can also be used to separate analytes that are in solutionwith the use of microfluidics and electrochemistry that are easilyco-manufactured. The novel substrate has a large effective surface areaand can be tailored to enhance additional optical detection techniquessuch as fluorescence. This enables the substrate to serve as a platformfor a variety of molecular diagnostics.

In vitro molecular diagnostics can be performed with a variety ofsensing modalities that measure optical, electrical, radioactive, ormass spectroscopic properties of the material under test (i.e., theanalyte). In most scenarios, the analyte is processed so it isselectively bonded to a compound in the apparatus. Sometimes either theanalyte or a mating compound is tagged with a label such as afluorophore, nanosphere, or another agent that is then detected toindicate the presence and/or concentration of the principal analyte oranalytes.

The present invention is, among other things, concerned with a photonicdiagnostic technique (e.g., Raman spectroscopy utilizing SurfaceEnhanced Raman Scattering, or SERS) that can be label-free and may ormay not require the analyte to be bonded to another compound.

Raman scattering is the process whereby an optical photon inelasticallyscatters off a molecule by coupling with the vibrational modes of themolecule. The scattered photon energy is reduced (Stokes) or augmented(anti-Stokes) by the energy of the vibrational mode. The Raman scatteredlight has a detailed spectrum that is essentially unique for biologicaland many other molecules as it encodes all of the bonds present in themolecule, and may indicate the conformation of the molecule as well.Raman spectroscopy (RS) is the technique whereby the spectrum ismeasured by quantitatively recording the Raman scattered light as afunction of wavelength or wavenumber (cm⁻¹) when a monochromatic (e.g.,a laser) beam illuminates the sample. An example of a Raman spectrum isshown in FIG. 1.

One barrier to the use of Raman spectroscopy is the small cross-sectionfor Raman scattering, a factor of ˜10¹⁴ less than the cross-section forfluorescence. This problem is mitigated when the molecule is adsorbedonto, or is near, a conductive surface with structure at the appropriatenanometer-sized scale. Then, the incident electromagnetic field, e.g.,the laser, the plasmon modes of the conduction electrons, and themolecular vibrational modes strongly couple and greatly enhance theRaman scattering cross-section. This electromagnetic (EM) enhancementcan increase the cross-section by up to a factor of 10¹⁴, locally, andby a factor of 10⁴-10⁸ averaged over the ensemble of molecules nearby oradsorbed on a conducting surface. In addition to the EM enhancement,additional enhancement can come from chemical interactions or from aresonance of the molecule with the input laser (i.e., the Raman pump)wavelength. The latter effect is usually termed Surface EnhancedResonance Raman Scattering (SERRS).

The basic correlation of nanoscale structure and surface plasmonexcitation is apparent from Mie scattering theory. The characteristicstructure size for effective SERS enhancement ranges from tens of nm forisolated metal particles to several hundred nm for nanostructuredsurfaces. The optimum feature size for maximum enhancement scales withthe pump wavelength. Both theoretical and experimental studies haveshown that the EM enhancement is maximized where particles are nearly oractually touching, or generally where the surface is discontinuous andelectric fields are presumed to be large. There is evidence thatperiodic structures increase the SERS enhancement. However, it isgenerally acknowledged that detailed knowledge and prediction of thesurface enhancement phenomenon is not completely understood at thistime.

Many substrates have been used for SERS. Initial work used metalelectrodes that were electrochemically etched to produce nanoscaleroughness. Those substrates were particularly unpredictable and oftenchanged their properties over time due to electrochemical reactions.Molecules in solution have been analyzed with SERS by introducingnano-sized metal particles into the solution. These particles haveincluded silver and other metal colloids and, more recently, nano-sizedspheres that are produced by a variety of means. Over the years, SERSsubstrates have generally been made by forming a nanostructure, thenevaporating a metalized layer onto the nanostructure. The underlyingsubstrate has included lithographically etched materials, chemicallyetched materials, and a self-assembled monolayer of plastic nanospheres.Additional techniques for forming SERS substrates involve evaporatingmetal films onto glass slides—this can include depositing metal islandson the glass slide by not uniformly covering the surface of the slide,and nanopatterning the surface of the slide by using the interstices ofa self-assembled nanosphere layer as apertures in a technique callednanosphere lithography. Recently, a substrate has been announced thatuses a metalized photonic crystal formed by semiconductor lithography.Another substrate with interesting properties, but which is probably notaffordably manufacturable, is e-beam lithography of silicon.

The interest in SERS as an analytical technique comes from severalfeatures. The signal from SERS can be larger than the fluorescencesignal due to the surface enhancement and the shorter lifetime of Ramanexcitation relative to fluorescence, which can enable more scatteringevents per molecule per unit time. The SERS spectrum is typically 10˜100times narrower than the typical fluorescence spectrum. This addressesone serious issue with fluorescence studies, i.e., the number ofdifferent labels that can be distinguished in the same assay. Withfluorescence labels, a maximum of 12 labels can be distinguished. Thisnumber can increase, significantly, if SERS-active labels are used. Atleast one company, Nanoplex Technologies, Inc. of Mountain View, Calif.,is focused on developing labeling nanoparticles that are SERS-active.

Moreover, the SERS spectrum is essentially unique for each analyte. Thisoffers the opportunity to identify specific analytes that do not need tobe labeled with a fluorescent dye. This same feature can also enablelabel-free binding detection. Many assays are designed to measure thebinding or conjugation of two complementary materials—e.g., aprotein-ligand, a DNA strand-oligonucleotide, etc. The SERS spectrum hasbeen shown by to exhibit variation that indicates when binding hasoccurred, as is seen in FIG. 2. SERS has been used to quantitativelydetect the amount of an analyte and measure time-varying signalsindicative of binding kinetics, as is seen in FIG. 3. For the case of aprotein (avidin) and a small molecule (biotin), a Raman spectralsignature of the bound complex shows variation in the protein spectrum(tryptophan bands) and the presence of a biotin peak at 690 cm⁻¹ whichhas information about the structure and function of the bound complex,as is seen in FIG. 4.

With the surface enhancement in SERS analyses, very small quantities ofmaterial can be detected using spatially-resolved detection. Someexperiments have achieved attomole sensitivity and, for isolatedparticles, detection of single molecules has been achieved. There havebeen numerous papers on SERS and related studies of plasmon resonancewith nanoscaled structures. There have been hundreds of papers per yearon this topic since 1995.

As many of the advantages of SERS have been known for years, and withthe level of interest in the literature, it is instructive to understandwhy SERS analysis has not become more prevalent. One major reason oftencited in the literature is the lack of availability of a suitablesubstrate material. Many substrates exhibit a wide variation insensitivity from realization to realization, or over time. Othersubstrates are expensive or difficult to prepare. The development ofquantitative analysis using SERS depends on the availability ofsubstrates that are relatively easy and inexpensive to produce, arereadily reproducible, and offer the potential to tailor the surfacefeatures for strong Raman scattering enhancement. While some of the SERSsubstrates I have described above meet one or more of these criteria, nosubstrate is currently commercially available that meets all of them.

SUMMARY OF THE INVENTION

In accordance with the present invention, a novel substrate is provided,a region of whose surface is pre-processed with a laser to yield ananometer-scaled structure and which has a metal film applied to thenanostructured region so as to form the novel substrate. In some cases,the substrate can comprise an appropriate metal so that thelaser-processing provides the nanometer-scaled metal structures desiredfor SERS applications—in these cases, the subsequent step of applying ametal film to the laser-processed surface may be omitted.

In one form of the invention, a means of introducing a cell or asolution or mixture (i.e., the analyte) to that nanostructured,metalized region is provided so that the analyte is either adsorbed ontothe region, or lies in solution covering the region, or is deposited asa powder on the region, or is a gaseous vapor adjacent to the region,whereby to facilitate SERS analysis of the analyte.

And in one form of the invention, a means of separating or fractionatingthe solution or mixture (i.e., the analyte) is provided.

And in another form of the invention, a means of introducing a reagentto the solution or mixture (i.e., the analyte) is provided.

A detection apparatus is provided consisting of (i) a light source toirradiate the nanostructured, metalized region (or a portion thereof)and the analyte, and (ii) an optical detector to sense the scatteredradiation. In particular, a narrowband laser and a spectrometer areprovided which determine the Surface Enhanced Raman Spectrum (SERS) ofthe analyte material near the nanostructured, metalized surface.

In one preferred form of the invention, there is provided a method forsensing at least one of the presence and quantity of an analyte, whereinthe method comprises:

providing a base that has been structured using laser processing so asto provide at least one patterned surface;

applying a metal to the at least one patterned surface so as to provideat least one metalized patterned surface; and

using the at least one metalized patterned surface as a substrate forperforming a diagnostic assay of the analyte.

In another form of the invention, there is provided an apparatus for usein performing a diagnostic assay of an analyte, the apparatuscomprising:

a base that has been structured using laser processing so as to provideat least one patterned surface; and

a metal applied to the at least one patterned surface so as to provideat least one metalized patterned surface.

In another form of the invention, there is provided a substrate forsensing at least one of the presence and quantity of an analyteintroduced to the substrate, the substrate comprising:

a base that has been structured using laser processing so as to provideat least two patterned surfaces; and

a metal applied to the at least two patterned surfaces so as to provideat least two metalized patterned surfaces.

In another form of the invention, there is provided a substrate forsensing at least one of the presence and quantity of an analyteintroduced to the substrate, the substrate comprising:

a base that has been structured using laser processing so as to provideat least one patterned surface; and

a metal applied to the at least one patterned surface so as to provideat least one metalized patterned surface;

wherein the base comprises at least one via for directing the analyteacross the at least one metalized patterned surface.

In another form of the invention, there is provided an apparatus forsensing at least one of the presence and quantity of an analyte, theapparatus comprising:

a base that has been structured using laser processing so as to provideat least one patterned surface;

a metal applied to the at least one patterned surface so as to provideat least one metalized patterned surface; and

an optical characterization module comprising:

-   -   a light source for directing light at an analyte disposed on the        at least one metalized patterned surface; and    -   a detector that measures the light scattered by the analyte.

In another form of the invention, there is provided a method for sensingat least one of the presence and quantity of an analyte, wherein themethod comprises:

providing a casting base that has been structured using laser processingso as to provide at least one casting base patterned surface;

duplicating the base by casting so as to provide a working base havingat least one working patterned surface;

applying a metal to the at least one working patterned surface so as toprovide at least one metalized patterned surface; and

using the at least one metalized patterned surface as a substrate forperforming a diagnostic assay of the analyte.

In another form of the invention, there is provided apparatus for use inperforming a diagnostic assay of an analyte, the apparatus comprising:

a base that has been structured using laser processing so as to provideat least one patterned surface by melting and resolidification of thebase, wherein the patterned surface is characterized by structuresranging in scale from 10 to 2000 nanometers and further wherein thepattern is stochastic in all three spatial dimensions; and

a metal applied to the at least one patterned surface so as to provideat least one metalized patterned surface.

In another form of the invention, there is provided an assemblycomprising:

a metal base that has been structured using laser processing so as toprovide at least one patterned surface by melting and resolidificationof the base, wherein the patterned surface is characterized bystructures ranging in scale from 10 to 2000 nanometers and furtherwherein the pattern is stochastic in all three spatial dimensions; and

an analyte disposed on the at least one patterned surface.

In another form of the invention, there is provided a method for sensingat least one of the presence and quantity of an analyte, wherein themethod comprises:

providing a base that has been structured using laser processing so asto provide at least one patterned surface characterized by structuresextending in all three spatial dimensions, wherein the laser processingcomprises the selective application of pulsed laser energy to the basewhereby to melt a surface layer of the base which resolidifies so as tocreate the structures of the at least one patterned surface, and furtherwherein the melting across the laser spot is non-uniform with finestructure on scales comparable to the resulting structures of thepatterned surface;

applying a metal to the at least one patterned surface so as to provideat least one metalized patterned surface, wherein the at least onemetalized patterned surface has a surface profile configured to providelarge electric fields when electromagnetic energy is delivered to the atleast one metalized patterned surface;

positioning the analyte on the at least one metalized patterned surface;and

performing a diagnostic assay of the analyte by deliveringelectromagnetic energy to the analyte and/or the at least one metalizedpatterned surface.

In another form of the invention, there is provided a method for sensingat least one of the presence and quantity of an analyte, wherein themethod comprises:

providing a base that has been structured using laser processing so asto provide at least one patterned surface characterized by structuresextending in all three spatial dimensions, wherein the laser processingcomprises the selective application of pulsed laser energy to the basewhereby to melt a surface layer of the base which resolidifies so as tocreate the structures of the at least one patterned surface, and furtherwherein the melting across the laser spot is non-uniform with finestructure on scales comparable to the resulting structures of thepatterned surface;

positioning the analyte on the at least one patterned surface; and

performing a diagnostic assay of the analyte by deliveringelectromagnetic energy to the analyte and/or the at least one patternedsurface.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other objects, features and advantages of the presentinvention will be more fully disclosed or rendered obvious by thefollowing detailed description of the preferred embodiments of theinvention, which is to be considered together with the accompanyingdrawings wherein like numbers refer to like parts, and further wherein:

FIG. 1 is a schematic view of an exemplary Raman spectrum using SERS;

FIG. 2 is a schematic view of a SERS spectra associated with label-freeantibody-antigen binding;

FIG. 3 is a schematic view of a time-varying (kinetic) SERS signal forglucose partitioning by a self-assembled monolayer;

FIG. 4 is a schematic view showing the Raman spectra of A) avidin, B)avidin-biotin, C) tryptophan, and D) biotin in the lyophilized form;

FIG. 5 is a schematic view of a nanostructured silicon surface formed byfemtosecond laser irradiation at two different magnifications;

FIG. 6 is a schematic view of a nanostructured silicon surface, whereinthe surface is shown as a function of the number of femtosecond laserpulses, wherein the entire surface is structured between 20-100 pulsesand additional pulses generally deepen the structures;

FIG. 7 is a schematic view of a stainless steel surface nanostructuredwith femtosecond laser pulses and micropatterned with a hexagonalmask—note that the structure near the mask boundary is partially orderedin rows while in the interior, the lateral structure of the surface isdisordered;

FIG. 8 is a schematic view of nanostructured metal surfaces formed byfemtosecond laser irradiation—the left image shows a titanium surfaceand the right image shows a copper surface, and the scale bar is 1 μm atleft, 5 μm at right;

FIG. 9 is a schematic view of nanowires formed on silicon when thefemtosecond laser processing is done with an organic solvent in place ofwater, which was used in the process shown in FIG. 5—the images of FIG.9 show a surface processed with methanol, with the left image having a20 μm scale bar, and the right image having a 2 μm scale bar, and beingpositioned at the edge of the region that was laser processed;

FIG. 10 is a schematic view of scanning electron micrographs of orderedsilicon spikes formed by masking the irradiated sample with: (a) and(b), a 30 μm hexagonal grid, and (c) and (d), a 20 μm square grid,wherein the nearly Gaussian spatial intensity profile of the laser pulseis shown at the top in grayscale (white corresponds to maximumintensity);

FIG. 11 is a schematic view of scanning electron micrographs of:(a)-(e), silicon spikes formed with a square grid after increasingnumber of laser pulses, wherein the direction of the electric field isvertical in (a)-(e), and with (f), spikes formed with the grid rotated45° relative to the grids in (a)-(e);

FIG. 12 is a schematic view of an exemplary device incorporatingmicrofluidic delivery of a reagent, and separation via electro-osmoticpressure, wherein analyte is injected through a port at the left, thevoltage is placed across the two electrodes to effect separation as ionsmigrate through the nanostructure, a reagent is injected at theappropriate time through the top port, and a noble metal film enablessurface enhanced detection, e.g., SERS, of a reaction product;

FIG. 13 is a schematic view of silicon nanostructured with femtosecondlaser pulses, wherein the pulse energy increases from (a) to (b), asdoes the spatial scale of the structure, and the scale bar in the insetin (a) is 1 μm; and

FIG. 14 is a schematic view showing a novel Raman spectroscopy systemfor performing a diagnostic assay on an analyte positioned on asubstrate having a metalized patterned surface.

It should be appreciated that the drawings are intended to provide abetter understanding of the present invention, but they are in no wayintended to limit the scope of the invention.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

A novel substrate that meets all of the criteria discussed above foreffecting surface enhancement of an analyte for SERS analyses is formedby a novel semiconductor or metal structure which includes nanopatternedsurfaces fabricated by laser processing using femtosecond lasers. Thefemtosecond laser irradiation of the semiconductor or metal structure(e.g., silicon) in the appropriate environment can produce a variety ofinteresting nanostructures. These nanostructures are either produced ina metal substrate or the nanostructured surface is subsequentlymetalized so as to form the complete substrate which can provide thedesired optical effect, e.g., for SERS applications.

One such structure is called black silicon, as micron-scale spikesformed on the surface of the silicon change the reflectivity of thesilicon so its absorptance is significantly increased in the visible,and well into the infrared, spectrum.

Another structure has nanoscale bumps or spikes when the base materialis covered with water, or another liquid, during laser processing.

Another member of this family of structures involves the irradiation ofmetal surfaces which can produce nanosized cylindrical asperities alongthe surface.

Yet another surface geometry which may be used in the present inventionis the growth of thin nanowires which can result when an organic solventcovers the surface, e.g., methanol over silicon, during laserprocessing.

These nanopatterned structures are shown in FIGS. 5-9.

While the dynamics of forming the desired nanopatterned surfacestructures using femtosecond lasers is not thoroughly understood at thistime, the basic explanation is that the energy deposition by thefemtosecond pulse excites surface waves in the base material.Diffraction by the surface structure further roughens the structure thatthen ‘freezes’ in place as the laser heating is removed.Non-uniformities induced in the laser beam before it is incident on thesurface can also play a role in the laser structuring process. Theeffect of multiple pulses can be seen in FIG. 6.

It will be appreciated that the laser structuring process involvesmelting and resolidification that rearranges the substrate material inthe desired manner. More particularly, this process produces a surfacestructure with random features in all 3 dimensions, both along the planeof the substrate and perpendicular to the plane of the substrate. Inother words, this laser structuring of the substrate provides astructured surface characterized by a stochastic pattern in all threespatial dimensions due to random aspects of the structuring process,with the laser-induced structures having the spatial characteristicsdesired for SERS applications.

Details for forming microstructured or nanostructured surfaces usingfemtosecond lasers are provided in:

(i) U.S. Provisional Patent Application Ser. No. 60/293,590, filed May25, 2001 for SYSTEM AND METHODS FOR LIGHT ABSORPTION AND FIELD EMISSIONUSING MICROSTRUCTURAL SILICON;

(ii) U.S. patent application Ser. No. 10/155,429, filed May 24, 2002 byJames E. Carey, Catherine Crouch, Claudia Wu, Rebecca Younkin, and EricMazur for SYSTEM AND METHODS FOR LIGHT ABSORPTION AND FIELD EMISSIONUSING MICROSTRUCTURAL SILICON;

(iii) U.S. patent application Ser. No. 10/950,230, filed Sep. 24, 2004by James E. Carey and Eric Mazur for SILICON-BASED VISIBLE ANDNEAR-INFRARED OPTOELECTRIC DEVICES;

(iv) U.S. patent application Ser. No. 10/950,248, filed Sep. 24, 2004 byJames E. Carey and Eric Mazur for MANUFACTURE OF SILICON-BASED DEVICESHAVING DISORDERED SULFUR-DOPED SURFACE LAYERS;

(v) U.S. patent application Ser. No. 11/196,929, filed Aug. 4, 2005 byEric Mazur and Mengyan Shen for FEMTOSECOND LASER-INDUCED FORMATION OFSUBMICROMETER SPIKES ON A SEMICONDUCTOR SUBSTRATE; and

(vi) International (PCT) Patent Application No. PCT/US05/34180, filedSep. 23, 2005 by Eric Mazur and James E. Carey for MANUFACTURE OFSILICON-BASED DEVICES HAVING DISORDERED SULFUR-DOPED SURFACE LAYERS.

The foregoing six patent applications are hereby incorporated herein byreference.

The nanopatterned surface structures may then be metalized by thedeposition of a noble metal film so as to form a novel substrate whichcan serve to effect the desired surface enhancement of an analyte foroptical analyses. More particularly, nanopatterned surface structures,after application of a noble metal film, satisfy all the criteria for aSERS substrate. Alternatively, the laser processing may be performed onan appropriate metal substrate, in which case the subsequentmetallization step may be omitted. The surface characteristics of thenanostructured material may be tailored by adjusting (i) the energy ofthe laser pulse, and (ii) the manner in which the pulse train isdelivered to the substrate. This allows one to tailor the nanostructuredsubstrate so that the final metalized surface has the right scales forSERS enhancement. The nanostructured surfaces are straightforward tofabricate with commercially-available femtosecond lasers. With a 20 Wfemtosecond laser, an area equivalent to an entire 25 mm×75 mmmicroscope slide can be fabricated in less than 2 minutes, so thismaterial can be manufactured at a reasonable cost. Due to the fact thatfemtosecond pulses deposit energy before it can diffuse, the energydeposition, and hence the statistical properties of the nanopatternedsurface, are predictable and reproducible if the laser pulses aredelivered in a controlled fashion.

As surface plasmon modes are excited when any metal is irradiated with alaser, it will be appreciated that any nanostructured metal surfaceexhibits some enhancement of the induced electric field. Thus, in somecases, useful substrates can be fabricated by laser patterning of ametal base without a subsequent metalization step. The enhancementfactor is likely to be greatest when a noble or coinage metal isstructured, e.g., gold, silver, or copper.

When laser patterning is performed, the surface of the material beingstructured is subject to intense electric fields, which can ionize thesurface material or the fluid which is in contact with the surface,whether it is a gas or liquid. This ionized material can be veryreactive and cause the surface material to undergo a chemical reaction.Consequently, a structured metal surface may not have a pristine layerof the metal at the surface, which will generally reduce the enhancementof any induced electric fields. In some cases, it may have a thin,oxidized layer.

Thus, in some situations, a subsequent step may be desirable or requiredto treat the structured metal surface when metal substrates are directlylaser-patterned without a subsequent metallization step. Moreparticularly, in order to form a useful surface, well-known chemical orelectrochemical processing may be employed to etch away or convert themodified surface layer into a pristine metal which will exhibit greaterenhancement. This chemical or electrochemical processing will vary withthe base material and the character of the surface structure inducedwith laser patterning.

Laser-nanostructured substrates have been demonstrated to provide auseful base for devices which incorporate subsequent metallization andcan be used as substrates for molecular detection using surface enhancedRaman spectroscopy (SERS). Substrates nanostructured by processing withan ultrafast (femtosecond) laser have been fabricated in silicon,stainless steel, titanium and copper. This processing has used 400 nmwavelength lasers with the substrate material in a water bath. The laserwavelength and aspects of the surrounding medium such as its refractiveindex and thermal properties have some influence on the details of thenanostructure. The resulting metal nanostructure should be tailored tothe particular Raman laser wavelength(s) which may be employed in theSERS application. It is generally desirable to produce columnarstructures which have a controlled, high-aspect ratio (height towidth/separation), since such columnar structures have been demonstrated(theoretically and experimentally) to produce large enhancement factors.

My previous disclosures have described irradiating an area of thesubstrate material with a laser spot. The workpiece and the laser spotare relatively translated (i.e., moved relative to one another) in orderto cover a larger area. The effective time that the spot dwells on aparticular spot affects the depth and details of the nanostructure thatis produced. For instance, a slower relative feed rate means that eachspot on the material “sees” more laser pulses. This has been shown toproduce deeper nanostructures with more pulses. Theabsorption/reflectivity of the material is a function of thenanostructure depth, among other factors, so there is an optimal depthfor a particular wavelength of Raman laser.

My previous disclosures have also described placing a mask over thesubstrate material. In general, these masks have holes with diametersmeasured in microns. This has the effect of forming arrays of structuredmaterial, and also affects the details of the nanostructure inside thestructured regions. For example, the boundary conditions of theillumination at the edge of a mask aperture appear to induce some orderin the nanostructure, at least in the direction perpendicular to theaperture boundary.

Among other things, the present disclosure introduces several new ideasthat may improve the speed or efficiency with which the surface isstructured. The goal is to produce nanopatterned surfaces with a givensurface morphology, i.e., spatial pattern of bumps, pillars or spikes.In the following discussion, I will address the formation ofnanopillars, but this is intended to be without loss of generality. Asthe laser melts a layer of material at the surface, which then flows andresolidifies, tailoring the portion of the surface that is melted couldimprove the speed or efficiency of that operation. For example, it ispossible that by only melting the portions of the surface which areintended to end up as troughs of the final surface, the pillars willform more quickly and the rate of processing of the surface couldincrease.

One way to accomplish this is to structure the laser light whichirradiates the substrate surface. For example, the laser light could besplit into two or more beams which are then recombined at an angle toform a fringe pattern or other pattern that has alternating light anddark regions. The melting will preferentially occur at the lightregions, which will presumably end up as the troughs of the surface,while the dark regions will melt less and will presumably end up as thepillars.

The laser beam can be split and recombined using well-knownbeam-splitters, mirrors, and lenses. However, since I generally want toproduce nanostructured regions, the various beams will have to combineat relative large angles which makes the required optics difficult toconstruct. In addition, the motion of the workpiece relative to thestructured light must leave the nanostructure surface aligned with thestructured light. Since this can require the motion to be periodic withnanometer periods, it can be expensive to accomplish. So, other ways ofmodifying the spatial melting of the substrate may be preferred.

Another way to pattern the melting by the laser is to structure theincident laser beam by passing it through an optical mask that has ananostructured pattern, e.g., a master mask with 100 nm diameter holes.For example, a chrome-on-glass mask can be nanopatterned with standardlithography techniques (either light-based or ion-based). The mask canthen be placed between the structuring laser beam and the substratematerial. The chrome material will generally reflect the laser, definingregions which are melted less, while the clear regions will pass thelaser, defining regions which are melted more. If the mask is fixedrelative to the substrate material, the melting pattern will remainfixed, avoiding the problem with structuring the light. Other masks,such as a thin metal mask, may be preferred. It will be appreciated thatthese thin metal masks have fine structure that is much smaller than thedimensions of the laser spot, thereby efficiently promoting the laserstructuring of the substrate on that fine scale.

This approach also has some technical difficulties, since the distancebetween the mask and the substrate material cannot be too large ordiffraction can spoil the intensity pattern defined by the mask. Thesmall distance will affect the volume of liquid which is present to coolthe substrate material, and may be evaporated by the structuring laser.It is possible to flow liquid in the gap between a mask and thesubstrate material, or use a mask thin enough so it can be placed on thesubstrate material. It is also possible to arrange the path which thestructuring laser makes across the substrate material, and to arrangethe flow path of the liquid, so the irradiated region is always coveredwith liquid.

Another way to impose a pattern on the material which is melted is byimprinting a pattern on the surface of the substrate material thataffects the local absorption of the incident laser radiation. One way todo that is to deposit a material that enhances either the absorption orreflection of the laser light. For example, using standardphotolithographic techniques, a thin layer of inorganic material can bedeposited on the substrate material. The substrate material is coatedwith photoresist which is then illuminated with the pattern. Dependingon whether negative or positive processing is used, the substrate can beprocessed so either the exposed or unexposed regions are left with theinorganic material, leaving a nanopattern. Nanoimprint lithography,using a nanostructured tool to print a patterned, thin layer of materiallike a printing press, is another cost-effective means of depositingmaterial that could seed the nanostructure formation.

Another way to cause non-uniform melting across the laser spot in orderto improve the efficiency of the laser processing is to deposit a layerof an inhomogeneous material on the surface which is to be structured.This deposited material ideally has inhomegeneities on nanometer scalesand is ideally applied in a thin layer that may consist of a monolayeror less, on average, of the material. It may be sprayed, spin-coated ordrip-coated onto the substrate. One example is the application of aself-assembling monolayer of plastic nanospheres, which will result in anon-uniform incident flux of the structuring laser onto the substrate.

It will be appreciated that the various methods of applying anon-uniform absorber or reflector to the substrate all require theapplied pattern to have fine structure that is much smaller than thedimensions of the laser spot, thereby efficiently promoting the laserstructuring of the substrate on that fine scale.

Another way to affect the local absorption is to do a preliminarystructuring of the surface with the processing laser and a mask. Here,the mask is only used initially to modify the surface in a way thatseeds the subsequent structuring, rather than keeping the mask in placeduring the entire structuring process. This may be more efficient in itsuse of the laser light, since the attenuating mask is only presentinitially, and it may prolong the lifetime of the mask.

The foregoing are some examples of ways to modify the absorption orreflection of the substrate material so that the structuring laser canproduce the resulting nanostructure more efficiently. They also offer away by which the morphology of the surface can be controlled, since theyobviously affect the transverse spatial pattern of the nanostructure. Itshould be appreciated that the foregoing examples are offered by way ofexample but not limitation.

With simple masks and appropriate delivery of the femtosecond pulsetrain, the surface of the substrate can be micropatterned with a seriesof nanopatterned regions. If the boundary conditions of the processedsurface are controlled, the resulting structure self-organizes into aquasi-periodic pattern. FIGS. 7-11 show examples of micropatternedregions that exhibit stochastic and quasi-periodic structures atdifferent scales and in different regions of the surface.

The ability to micropattern the surface of the base material (e.g., asemiconductor or metal material) and do other laser machining isparticularly interesting for the application of this novel substrate toarrays. Microarrayed patterns of nanostructures can be fabricated thatwill allow these substrates to be used in a manner similar tomicroarrays that are used for genomic and proteomic screening. Forscenarios where specific detection of molecular species can be performedwithout labeling the molecules, as must be done for fluorescencedetection, the resulting arrays can have greater specificity andsensitivity than conventional microarrays and eliminate the need to doPCR replication, which also adds noise.

Other desirable features relating to microfluidics and electrochemistrycan easily be co-manufactured and add additional functionality to thenovel substrate. For example, the same femtosecond laser used tonanostructure the surface can also be used to ablate material andmachine micro- and nano-vias in the substrate. Adding a cover to thesubstrate forms channels that can be used to direct fluid flow acrossthe substrate. This can be accomplished by placing a flat cover materialon the substrate and allowing liquid surface tension to hold the coverin place. In addition, the cover material can be glued or thermallybonded to the base material. One possible material for the cover,commonly used in micro- and nano-fluidic devices, ispolydimethylsiloxane (PDMS).

A unique advantage of this approach for forming a SERS substrate is theability to make deep or high-aspect nanostructures that are preciselytailored. No other approach, except perhaps porous silicon produced byetching, can affordably make similarly convoluted structures, and thosestructures are poorly controlled. One benefit of my substrate is thatthe effective surface area of the substrate is much larger than theplanar area of the substrate. This greatly increases the volume ofanalyte that can be placed in close proximity to the metal filmsdeposited on the nanostructures and supporting the plasmon modes, andthus increases the dynamic range of the measurement.

When the nanostructured region is covered with a cap or other cover, theresult is a nanoporous channel. This feature can be used to separate orfractionate different materials that are introduced via a solution ormixture to the substrate. The separation can occur as the result ofdifferential flow resistance to fluid pressure or as the solutes ormixture components are subjected to electro-osmotic pressure by applyinga voltage across a non-conducting substrate, or can occur due toelectrophoresis of ionic species in solution.

A flow of analyte across the substrate can also be coupled withfunctionalization of the substrate to enable more complex detection. Forexample, an analyte mixture flowed over the substrate could possiblycontain different compounds which have different chemical properties.Different regions on the substrate could be coated with differentmaterials that preferentially attract or repel certain compounds.Examples of such coatings could be oligonucleotides, proteins, orantibodies to which specific molecules can bind. They can also becoatings which, for example, attract or repel molecules with specificchemical groups, ionic charges, or pH values. The regions on thesubstrate can be ordered along the direction of the flow, perpendicularto the flow, or in a matrix which is correlated with a complex flowpattern that may involve movement of solution or solute in multipledirections. Spatially variable functionalization of the substrate andmaterial flow are coupled with time-varying optical, e.g., Ramanspectral measurements, in order to detect particular analytes that matcha spatial or temporal pattern that depends on the functionalization.

It should be appreciated that a metalization step may be necessary inorder to introduce the thin noble metal film onto the laser-generatednanostructures. As a result, metalization for other purposes can beeffected without significantly complicating the substrate fabricationprocedure. Thus, for example, by placing a mask over the substratebefore metalization, electrodes can be patterned on the substrate. Thisallows a voltage to be applied along a channel that is formed in thesubstrate which will cause various ions in the solution or mixture tomigrate along the channel at rates that depend on the size of the ionsand the porosity of the channel. These electrodes also enable otherelectrochemical reactions to be driven.

An example of a complex structure involving microfluidics andelectrochemistry is shown in FIG. 12.

Other features of the substrate can control how an analyte, introducedto the substrate in liquid form, is moved or held in place. Forinstance, the surface of the substrate can be machined such thatstructures are formed that confine a small liquid drop which can bespotted onto the surface. In addition, the surface of the substrate canbe coated with various materials that either promote or inhibit adhesionand adsorption onto the surface. A common technique to promote adhesionis to apply a self-assembled monolayer of molecules with a thiol groupat the end. If the thiol group is added to the end of a molecule thatbinds with the analyte of interest, the analyte will be bound to thesurface.

Surface composition and chemistry plays a number of roles in using thesenovel substrates. Surface composition and chemistry can protect thesurface and affect the durability and shelf life of the substrates. Andsurface composition and chemistry affects whether the analyte adsorbsonto the substrate, thereby enabling the SERS effect. Surfacecomposition and chemistry also affects how the various conjugatetargets—genomic, proteomic, and other biomolecules—can be bound to thesurface. Surface composition and chemistry can also affect how materialintroduced to the substrate is confined.

It is desirable that the substrate properties for surface enhancement bestable over time. It is also desirable that the substrate not besusceptible to being attacked by a solution that is introduced foranalysis. While no material remains pristine forever, it is generallynecessary to inhibit oxidation of the metal film, especially if silveris used. It is also desirable to minimize any reaction of the analyte orsolution with the substrate, since that could change the behavior ofmolecules in solution and the coupling with the plasmon modes of themetal film. Since many analytes of interest are presented in a salinesolution, the surface should resist corrosion by salt water.

One solution is to protect the surface of the substrate with a very thinovercoat that inhibits chemical reactions but is thin and transparentenough that it does not change the optical properties and the ability ofmolecules to get close to the metal film. There are at least threedifferent approaches to overcoating the surface.

First, it is possible to add a thin overcoat of glass (e.g., silicondioxide) to the substrate. Depositing a thin film of silicon using avapor deposition process, followed by oxidation, can produce this layer.This approach has the advantage that the protective layer is glass, forwhich almost any subsequent functionalization that is required hasalready been developed.

A second approach is to cover the nanopatterned surface of the substratewith a self-assembled monolayer (SAM). Most often thiols, these SAMs areshort (several nm in length) linear molecules for which the thiol groupreadily attaches to metals such as gold. There are numerous literatureexamples for SAM attachment to silver and other metals as well. Thisapproach has the advantage that molecules can be used with functionalgroups that bind to target molecules which are used to functionalize thesurface. There can be some concern that a SAM on a convoluted surfacewill not cover uniformly and will have defects. However, as long as thedefects in the monolayer are a small fraction of the total area, thisshould not be a problem. Since the SAMs are deposited from a liquid,there is less concern that regions that are hard to reach will becovered. This is my preferred approach for overcoating nanopatternedsurfaces of the substrate.

This approach has been used by other researchers to functionalizesurfaces used for SERS substrates. For example, SAMS have been used tofunctionalize surfaces so that glucose could be partitioned fromsolution in order to attach enough material to give a significantlylarge SERS signal.

The SAMs must be chosen for stability, both with respect to degradationbefore the substrate is used and also with respect to use with analyteand washing solutions.

A third approach for overcoating the nanopatterned surfaces of thesubstrate is to apply a very thin parylene coating. Parylene is asubstance deposited by gas-phase polymerization which process is able touniformly cover surfaces with a pinhole-free coating. However, thedesired thickness for the SAMs, tens of nm at most, is thinner than mostparylene coatings and it may require new engineering to apply theparylene coating uniformly.

Another aspect of protecting the nanopatterned and metalized surface isto keep it pristine until used. Note that in addition to the coatingsthat may be applied, the substrates will generally be stored and shippedin inert atmospheres such as nitrogen or argon.

The ability of various analyte molecules to adsorb onto thenanopatterned surface and to coat the surface with target moleculesdepends on the compatibility and affinity of the two materials (i.e.,the analyte material and the substrate material), and whether thesubstrate can be wetted. There is also an issue of non-specificadsorption by analytes in a mixture. Although the substrates should befunctionalized with as specific an agent as possible, the SERS spectrumcould provide sufficient information to look for the presence of thetarget analytes even if other analytes adsorb. This can become a matterof signal-to-noise, where finding a molecule that is a small fraction ofa mixture is more difficult than one that is the dominant species.

Minimizing non-specific adsorption will improve the signal-to-noiseratio. It may be accomplished by a combination of functionalization andthe use of blocking agents. For example, if a certain protein is thetarget molecule, the surface might be functionalized with a SAM to whichthe protein adheres. After the protein is bound to the surface, it maybe useful to block any remaining surface with a neutral agent that doesnot affect the part of the SERS spectrum used to measure binding. Milkprotein and Human Serum Albumin (HSA) have previously been used for thispurpose with other SERS substrates.

I discuss in general terms how each of the protective coatings could becoated with materials with the desired properties. If the metal filmsare overcoated with glass, then there are numerous commercial agents andprocesses that can be used to functionalize them so biomolecules ofinterest will adsorb or adhere to them. For example, Gelest, Inc. ofMorrisville, Pa. sells silane agents that are used to functionalizeglass slides for DNA, proteins, or cells.

If the metal films (overcoating the nanopatterned structures of thesubstrate) themselves are overcoated with SAMs, the SAM molecule must bechosen so the free end (not attached to the metal) has the rightfunctional group attached, i.e., an aldehyde, amine, or carboxyl group.Dojindo Molecular Technologies, Inc. of Gaithersburg, Md., is acommercial vendor of SAMs which can be deposited onto the metalizedlayer.

If a parylene coating is used, the polymer must first be oxidized beforea functional group can be added. This can be done in a weak air plasma,after which the desired receptors can be added.

In general, the analyte is introduced to the substrate in liquid form.Consequently, how the analyte is confined to a region on the substrateis determined by how liquids interact with the surface of the substrate.The liquid should wet the region where measurements will be made. It maybe helpful to have boundary regions that do not wet around these wettedareas so as to confine the liquid.

This is particularly important when using the substrates as microarrays.It is possible to use a combination of (i) micromachining and (ii)tailoring the surface wetting properties, to direct drops of spottedsolution to remain in a particular region so a large array ofexperiments can be performed without cross-contamination. There areseveral approaches to confining the spotted material.

The first approach involves the generation of selective surfaceroughness to confine spotted drops. In general, flatter surfaces arewetted more easily than rough ones. It is possible to pattern an arraywith two different levels of surface roughness using the laserprocessing I have discussed above. The active part of the array wouldcomprise isolated small regions with laser processed nanostructuresappropriate for SERS. The inactive (or confining) part of the arraywould be the intervening grid pattern. This inactive region could belaser processed to produce microstructured black silicon so that it ismuch rougher than the neighboring nanostructured material. Thisstructuring would ensure that a drop would preferentially wet thenanostructured region with its smaller roughness.

A second approach involves patterning the surface with differentchemical treatments. One example of a process for producing ahydrophobic grid surrounding isolated regions is as follows. A siliconwafer is coated with Teflon or another hydrophobic agent. A metal maskwith small (e.g., 50 micron square) holes can be placed over the wafer.The femtosecond laser is used to first ablate the hydrophobic agent fromthe small squares, and subsequently to form the SERS nanostructures.With the mask still in place, the nanostructured regions are metalizedand any further surface treatment is done. Then the mask is removed,leaving the hydrophobic agent under the masked pattern.

The greatly increased surface area of the substrate is another featureof the invention that offers advantages over competing approaches. Thenanostructured material has a much greater surface area than a similarsized flat surface. This enables greater amounts and concentrations ofreagents or binding materials to be deposited when the surface isfunctionalized and increases the resulting reaction rates and/orfractions of material that participate in the reaction.

A variety of techniques can be used to introduce analyte to thesubstrate in solid, liquid, or gaseous form. Some examples are to spot adrop onto the substrate, either manually or robotically, and allow thesolvent to evaporate. Alternatively, a cover slip could be placed overthe drop spotted onto the substrate to analyze molecules in solution. Ifa cover with a fluid port is bonded onto the substrate, the liquid couldbe pumped onto the substrate in an example of a microfluidic system. Asolid can be sprayed onto the substrate, or introduced as a suspension,after which the suspending liquid or gel can be evaporated orsublimated. The substrate can be placed in an atmosphere having analytein a gaseous vapor.

The concentration of analyte introduced in solid, liquid, or gaseousform can be increased by various means before the analyte is introducedto the substrate, e.g., by filtering a solid, liquid, or gaseousmixture. The concentration can also be increased by coating thesubstrate with a second material which preferentially attracts theanalyte under test.

Once the analyte is introduced, the Raman spectrum can be recorded byirradiating the analyte with narrowband (e.g., laser) radiation. Theincident radiation is usually focused on the sample and scatteredradiation is usually collected with the same lens. An optical systemwith a dichroic beamsplitter can steer the scattered radiation out ofthe optical path taken by the incident radiation and to a spectroscope.See, for example, FIG. 14, which shows a Raman spectroscopy system forperforming a diagnostic assay on an analyte positioned on a substratehaving a metalized patterned surface. The resulting SERS spectrum thatis collected can be analyzed in ways well known in the art to indicatewhat material is present and in what quantities. If the SERScross-section is large enough, and the Raman spectrum has distinguishingfeatures, this can be done with unlabeled molecules. If the amount ofmaterial is small, or its Raman spectrum is weak, the signal could beenhanced by binding another molecule that is labeled with an intenselyphotoactive dye prior to evaporation. If the sample could containseveral molecules, several different labeled markers with distinctspectra could be used.

In order to work with the microarrays, the capability to dispense verysmall volumes of material is required. This technology is available,off-the-shelf, using piezoelectric dispensing equipment such as thePiezoarray from PerkinElmer, Inc. of Wellesley, Mass. for spottingpicoliters (a 10 micron cube) or less.

A more involved detection starts with microarrays that arefunctionalized with various compounds such as unlabeled oligonucleotidesor proteins or other ligands. These can be bound to the surface with avariety of techniques, including covalent bonds to the self-assembledmonolayers previously described. Due to the specificity of the Ramanspectrum, some assays can use non-specific functional conjugates, unlikefor fluorescence assays. The analyte is introduced to the microarray andbinding to the ligand is facilitated by one or more variables includingtime, temperature, or other factors. Unbound material is optionallywashed away. Afterwards, a robotic Raman microscope then scans thearray, much as current readers analyze genetic microarrays. Thedifference is that the recorded Raman spectrum would indicate not onlythe amount of bound material, but also what the material was. Since morethan one material could be present in a given array location, atechnique such as Principal Component Analysis (PCA) would be used toestimate which materials are present and their amounts. Depending on theamount of material and/or the Raman signal strength, either thefunctionalized substrate or the analyte could be labeled with one ormore photoactive dyes.

Raman (and related IR) spectra cannot generally be used to identify themolecular structure of an unknown molecule, although some spectralfeatures can be correlated to specific bonds or functional groups.However, the spectrum in total is a linear sum of all the Ramanscattered light and can be analyzed for the presence and quantity of aparticular compound. For simple molecular spectra and/or uncomplicatedadmixtures, measurements often use a single spectral peak characteristicof a compound that may be present, typically by fitting a peak at agiven wavenumber with a simple profile that reflects the instrumentresponse (typically Gaussian). For more complicated measured spectra,either due to complex molecular spectra or admixtures of differentcompounds, more complicated eigenmodes can be used to analyze thespectrum. In spectroscopy, Principal Component Analysis (PCA) is oftenused to quantify admixtures. In this approach, eigenvectors of thecovariance matrix with large eigenvalues form a basis set used todeconvolve the measured spectrum using standard linear algebraicoperations. With this technique, cross-correlations of the differentmolecular spectra, statistical frequency of the analytes, instrumentresponse and noise characteristics can all be used to form an optimalestimate of the molecules present in a mixture and their concentrations.

Another potential application is in cell studies for signaling andexpression of various proteins. In one application, the cells wouldeventually be plated and fixed onto a coarse array. Markers labeled withphotosensitive dyes would then be washed with, and bound to, the targetmolecules. The cell membrane would be disintegrated and the cellularmaterial would drop onto the substrate. A spatially-resolved Ramanmicroscope could then detect the presence of the labeled markers withsome correspondence to location in the cell.

Another possible application is use of the substrate for in-vivocellular analysis. It is possible that suitably sharp nanospikes couldserve as a bed of Raman-enhancing nails that could produce SERS signalsfrom dye-labeled markers in live cells.

In order to perform dynamic assays, where the kinetics of binding aremeasured, the measurement device generally requires the capability toflow solutions over the nanostructured substrates. In some cases,library compounds will be flowed over substrates to which a targetmolecule is attached. This generally requires a microfluidic flow cell.The main requirement for this flow cell is that it must have anoptically transparent window so the Raman sensing is not inhibited bythe flow cell. Flow cells with channels that are laser machined inglass, as well as structures that use transparent polymers such asPolyDimethylSiloxane (PDMS) from which microfluidic structures caneasily be fabricated. The signal levels must be matched to themeasurement times so the integration times, which typically range fromunder 1 second to tens of seconds, can resolve the kinetics.

In order to achieve high throughput screening, several aspects of theinstrumentation require optimization. This generally requires that therobotics move fast enough to keep any delays due to translation of themeasurement apparatus relative to the array positions short compared tomeasurement times. Since the distances involved are quite small, this isgenerally not an issue for reasonable robotics (which can perform at therequired accuracy with speeds of meters/second).

The most critical aspect is the multiplexing of detection capabilities.It is desirable to multiplex multiple spectral measurements on a singledetector. The spectrometers used for Raman spectroscopy typically use adispersive element, i.e., a grating. Typically, the grating isilluminated with a slit. If the illumination along the length of theslit comes from different sources that do not overlap, the spectrum willconsist of multiple spectral strips from the different illuminationsources.

Charge Coupled Devices (CCDs) for spectroscopy are available withmillions of pixels and many are available with rectangular geometries.Depending on the number of multiplexed channels, a 1024×1024 or 1340×400CCD, like those available from Princeton Instruments, Inc. of Trenton,N.J. may be a good choice. Multi-channel spectroscopy is well-known tothe art—astronomers have been using multiplexed spectroscopes for manyyears, both ones that are fed with multiple discrete fibers and thosethat are imaging spectroscopes that can measure the spectrum of a linearimage.

Another aspect of the multiplexing is the laser source. Lasers used forRaman spectroscopy generally require high spectral purity. Currently,lasers for Raman spectroscopy are available from multiple manufacturerswith >350 mW of laser power. This should provide power for 10-100multiplexed measurements in order to meet high-speed requirements.

There are engineering tradeoffs with systems that either image themicroarray onto the sensor array or use discrete optical channels, e.g.,multiple optical fibers. Among other things, there are tradeoffs ofsimplicity (separate fibers) versus packing density and opticalthroughput (reimaged microarray).

A performance goal of interest to the pharmaceutical industry is 100,000measurements per instrument per day. This parameter is a familiarbenchmark to pharmaceutical industry personnel doing lead discovery. Fora nominal ten second measurement per array position, this benchmarkwould require 12 detection channels in parallel, assuming no time forthe robotic motion. It can be achieved by using multiple fibers, and thecorresponding instruments can take advantage of numerous opticalcomponents that have been developed for telecom applications that canhandle multiple fiber inputs and outputs and do so with very regularpositioning so no alignment is required.

There are other novel substrates being used for molecular analysis usingRaman spectroscopy and SERS. Tienta Sciences, Inc. of Indiana offersTeflon coated substrates which concentrate solutions via surface tensionand is working on adaptive silver films formed by controlled evaporateddeposition for SERS. Mesophotonics Limited of Southampton, Hampshire,UK, is offering photonic crystal material for SERS. The approachdescribed herein has significant advantages over the Tienta Sciences andMesophotonics substrates, especially for microarrays. My approach is lowcost while still retaining great flexibility to co-manufacturemicroarrays, microfluidic, and electrochemical features. My approachalso has much greater control, through the laser pulse energy andpolarization, than a controlled evaporation apparatus or aphotolithography and etching process can economically achieve. Theability to do laser machining of the substrates adds a dimension thatenhances the capabilities of my arrayed substrates. While photoniccrystals offer good reproducibility and potential for microarrayssimilar to my approach, they are more expensive to manufacture.Moreover, the reproducibility relies on controlling a photolithographyand etching process. Many manufacturers of MEMS devices have found thatthis can be costly to achieve in practice.

The approach described herein uses substrates with micro- andnanostructured surfaces created using femtosecond lasers. Thefemtosecond pulse deposits energy in the material faster than it canthermally diffuse. This gives rise to a variety of effects not possiblewith longer pulse laser processing. In the case of semiconductor andmetal surfaces, it leads to formation of spikes. When the surface isprocessed in a gas, which may react with the substrate material, micronsized spikes with large aspect ratios (height/base) are formed. When thesurface is processed in water or another liquid, nanometer-sizedfeatures are formed such as the remarkable surface structures shown inFIGS. 5-12.

FIG. 5 shows a silicon surface when the laser is normally incident tothe surface. Here, the nanopatterned surface is an assembly ofnanospikes. By changing the material, or the polarization and angle ofthe laser, a variety of different structures and scales can be formed.FIG. 8 shows the surface of two metals, titanium and copper, subject tothe same laser processing. The titanium surface forms quasi-cylindricalridges. The copper surface forms a less ordered assembly of nanospikes.

The details of the laser nanopatterning process are not currentlycompletely understood. However, it is generally thought that the laserexcites a surface wave in the material, which propagates and is thenfrozen when the surface cools. Random variations in the melting of thesurface layer, possibly due to asymmetry of the laser beam incident onthe surface or irregularities in the surface material, has a stochasticeffect after multiple laser pulses that produces the random surface withspiky features in silicon. This is seen in FIG. 6, where the progressionof the surface, after increasing number of pulses, is shown.

There is a threshold which must be achieved in order for the nanoscalesurface structuring to occur. Though it depends somewhat on the laserwavelength and base material, about 10 kJ/m² is required to excite therippling process. In order to produce a surface with fully developedstructure, a minimum number of pulses is required, on the order of 100.These two numbers combine to yield the surface area that can beprocessed per unit time as a function of average laser power, namely 1mm²/s per Watt of average laser power.

This number is encouraging. While femtosecond lasers are notinexpensive, they certainly cost less than the equipment necessary to dosemiconductor lithography on nanometer scales. The laser processing ismuch simpler than the various coating, exposure, and etching stepsrequired when fabricating nanostructures lithographically. Consequently,these substrates will be less expensive to manufacture.

These materials can be micropatterned very easily. FIGS. 7-11 showmicro- and nanostructured surfaces that have also been micropatterned.The patterning requires that a mask be placed on the surface of thematerial. The mask boundaries then naturally impress a pattern on theunderlying micro- or nanostructure. Micropatterning can facilitate theefficient partition of the substrate into an array whereby the surfacecan be functionalized with an array of targets, similar to themicroarrays used for fluorescence detection of oligonucleotide and otherbinding events. The surface structure can help confine analyte spottedonto the substrates.

The details of the structure inside the clear areas of the mask can showorder as well. The structure can self-organize either due to theboundary condition imposed by the mask on the laser excitation, or dueto the polarization of the laser, or a combination of the two. Thisproperty is of interest for SERS substrates since it is known thatordered structures can exhibit higher spectral selectivity or EMenhancement.

The statistical properties of the resulting surface are controlled bythe parameters of the laser excitation. See, for example, FIG. 11, wherethe polarization vector, in combination with a mask, affects the orderof the black silicon spikes. In FIG. 13, the lateral spatial scale ofthe structure is shown to be a function of the laser fluence per pulse.Larger fluence corresponds to larger structures. Similarly, the depth ofthe structures is a function of the fluence. Angling the surfacerelative to the laser also can help control both the fluence and theinfluence of the polarization vector on the symmetry of the surface(linear structures versus perpendicular spikes). These parameters enableus to tune the parameters of the surface, which in turn tunes theplasmon resonance that governs the coupling between the molecularvibrational modes and the laser light. In addition, the parameters ofthe surface structure can affect the conformational parameters of somemolecules when they are adsorbed.

The nanostructured surfaces are produced by laser processing withfemtosecond lasers. The details of these surfaces depend on a variety offactors. One is the environment surrounding the material when it isprocessed, namely the physical conditions. This can include temperatureand pressure, in the case of a material processed in a gaseousenvironment. It can also include a transparent liquid that is coveringthe material when it is irradiated.

The laser processing itself offers many variables that affect theresulting nanostructure. First is the pulse energy. As the laser pulseenergy is increased, the scale, both lateral and vertical, of theinduced structure changes. For low energies, the ripples are smaller inboth dimensions than for higher energies. It is well-known that thescale of the ripples affects the excitation of the surface plasmons bythe laser, with spatial scales of ˜200 nm giving good excitation withnear IR wavelengths and smaller scales working better with shorterwavelengths. Thus, the pulse energy is a useful control to match thesurface plasmon modes to the laser frequency.

Another variable is the material that is chosen. Possibly due todiffering laser absorption and thermal properties, the aspect ratio ofthe nanostructures that are produced are very different for differentmaterials. Silicon forms rather cylindrical features, while titaniumforms smaller bumps and copper is somewhere in between.

It is possible to generate cylindrical structures where the cylinderaxis is parallel to the surface, depending on the polarization of theinput laser. These structures are predicted to have high enhancement atthe boundary between cylinders where molecules may migrate, as thesolvent dissolves.

All of these variables will affect how the resulting SERS enhancementvaries with the morphology of the nanopatterned surface. The roughnessof the nanopatterned surface will have some effect on how easy it is tofunctionalize for various reactions. The symmetry (i.e., whether thereis a linear structure) will affect whether the polarization issignificant and may affect molecular conformation.

Other features of the invention include the patterning of the substratematerial. A micropattern can be created using a transmission mask, aswere the patterned surfaces shown in the Figures. It is also possible tomicropattern the surface by irradiating selected areas of the substratewith the laser. A laser spot focused to 25-50 microns can produce astructured region ˜50 microns in diameter that is isolated from aneighboring structure 100-200 microns away. This density is of interestto biochip microarrays, especially when one considers that theselectivity of the Raman spectrum may allow some analysis to beperformed with fewer array locations. These microarrays produced withouta mask will have different operational and economic characteristics.They may cost less to manufacture, but the boundaries of thenanostructured regions may not be as sharp, and they will not exhibitthe self-organized structures seen with transmission masks.

In addition to manufacturing the nanostructured surface directly bylaser processing, a nanostructured surface may serve as a master fromwhich nanostructured surfaces can be molded, or from which anintermediate negative master may be generated. A negative copy of thesurface can be made by casting PDMS or another flexible material againstthe surface, which can reproduce nanostructured features of the surfacewith excellent fidelity. Although the resulting castings are moredifficult to metalize than a semiconductor or metal, they are veryinexpensive to fabricate.

In addition to the laser processing, other features of the substratesthat will change their performance will depend on how the nanopatternedsurfaces are metalized. The metalization material, whether silver, orgold, or another metal, will change both the SERS effect and thechemistry of the surface. The plasmon resonances are different fordifferent metals. The chemistry may lead to different requirements whenthe surfaces are functionalized with oligonucleotides and ligands.

The properties of the metalization layer will also affect performance,depending on how the metalization is applied. One means of performingthe metalization is by physical vapor deposition performed byevaporating the metalization material. The detailed structure of themetalization layer and the resulting SERS performance will varydepending on whether the evaporation is done by resistive heating, ionbeams, sputtering, or other evaporation techniques. The metalizationlayer can also be applied by chemical or electrolytic deposition.

Another aspect to the metalization is control over the film geometry.Unlike many other metalized surfaces that have been used for SERS, someof the ones shown in the Figures are quite far from planar surfaces,either due to pillar-like structures or overall roughness. Consequently,the geometry of the evaporation will have a greater effect on the metalfilm that is deposited. One can induce a preferred axis by angling thesubstrate relative to the metalization source. Conversely, one may seedifferent surface enhancement when the film on the sides of pillar-likestructures is comparable to, or less than, the film on the tips.

The stability of the substrates over time will depend on how they arefabricated and stored. As the metalization layer may be reactive, sealedpackaging is generally required to preserve the substrates over time.

The spectroscope used to detect Raman scattering also affects thesensitivity of the diagnostic. While commercial systems are availablefor as little as $10,000, without a microscope, more expensive systemswill produce sharper spectra and will be more sensitive. Components andsystems are available from multiple vendors such as InPhotonics, Inc. ofNorwood, Mass. and Ocean Optics, Inc. of Dunedin, Fla., and lasers fromvendors such as Coherent, Inc. of Santa Clara, Calif., and opticalcomponents are available from various component and filter vendors andwill affect the system performance. The Raman spectrum can be measuredusing different illumination wavelengths and will interact with thesurface characteristics vis-à-vis performance.

A variety of assays are possible with the apparatus that I havedescribed. Similar assays have been performed with other SERSsubstrates. They include label-free detection of a biomolecule. Oneexample relates to Anthrax. Another example is the label-freedifferentiation of human insulin and insulin lispro.

Another possible assay uses an array of genomic material. If amicroarray is functionalized with various Raman labeledoligonucleotides, unknown genetic material can be introduced to reactwith the functionalized material and the unbound material washed away.The array, now with some spots including bound material and some spotswithout, is then scanned with the Raman spectrometer. The resulting dataindicates where the unknown material bound with the spotted material andin what quantities.

Another possible assay involves proteomics. An example is to look forbiotin-streptavidin binding. The substrate is functionalized with aligand such as streptavidin. Unknown material, possibly containingbiotin, which may be labeled with a dye such as Cy5, is reacted with thefunctionalized substrate and washed. Measurement of the resulting Ramanspectrum indicates whether the unknown material contained the conjugateprotein and in what quantities.

Another possible assay involves in-vivo cellular studies using SERS.This type of assay has been performed by injecting nano-sized metalparticles into live cells. The injected particles serve as the plasmonresonators for SERS. One disadvantage of this process is that it isinvasive to the cell. An in-vivo cellular assay using the substratedescribed herein relies on the spiky nature of the substrate to produceenhancement of the Raman signal even though the cell membrane liesbetween the substrate and the analyte within the cell. The enhancementfactor will be reduced, due to the nanometer distance from the substrateto material within the cell, so this assay may not be as sensitive asone where the cell was killed and the membrane disintegrated.

Although various exemplary embodiments of the invention have beendisclosed, it should be apparent to those skilled in the art thatvarious changes and modifications can be made which will achieve some ofthe advantages of the invention without departing from the true scope ofthe invention. For example, other assays are possible that do not useSERS. There are several photonic effects that employ the interaction ofelectromagnetic fields, surface plasmons and molecules, of which SERS isonly one example, that are possible with the substrates describedherein. These effects and the corresponding assays and all such variousembodiments, changes and modifications are to be understood to be withinthe scope of the present invention as described herein and as claimed inany appended claims.

1. Apparatus for use in performing a diagnostic assay of an analyte, theapparatus comprising: a base comprising at least one patterned surface,wherein the at least one patterned surface is characterized bystructures ranging in scale from 10 to 2000 nanometers in all threespatial dimensions and further wherein the pattern is stochastic in allthree spatial dimensions; the at least one patterned surface beingcreated from a pre-patterned surface on the base by a laser process,wherein the resulting patterned surface has elevations both above andbelow the elevation of the pre-patterned surface; and a metal applied tothe at least one patterned surface so as to provide at least onemetalized patterned surface.
 2. Apparatus according to claim 1 whereinthe structures are selected from the group consisting of bumps, pillars,spikes, fissures and holes.
 3. Apparatus according to claim 1 furthercomprising an optical characterization module comprising: a light sourcefor directing light at an analyte disposed on the at least one metalizedpatterned surface; and a detector for measuring light scattered by theanalyte.
 4. Apparatus according to claim 1 wherein the base furthercomprises at least one unpatterned surface, with the structures of thepatterned surface having elevations both above and below the elevationof the unpatterned surface.
 5. An assembly comprising: a metal basecomprising at least one patterned surface, wherein the at least onepatterned surface is characterized by structures ranging in scale from10 to 2000 nanometers in all three spatial dimensions and furtherwherein the pattern is stochastic in all three spatial dimensions; theat least one patterned surface being created from a pre-patternedsurface on the base by a laser process, wherein the resulting patternedsurface has elevations both above and below the elevation of thepre-patterned surface; and an analyte disposed on the at least onepatterned surface.
 6. An assembly according to claim 5 wherein thestructures are selected from the group consisting of bumps, pillars,spikes, fissures and holes.
 7. An assembly according to claim 5 furthercomprising an optical characterization module comprising: a light sourcefor directing light at the analyte disposed on the at least onepatterned surface; and a detector for measuring light scattered by theanalyte.
 8. A method for sensing at least one of the presence andquantity of an analyte, wherein the method comprises: providing a basecomprising at least one patterned surface, wherein the at least onepatterned surface is characterized by structures ranging in scale from10 to 2000 nanometers in all three spatial dimensions and furtherwherein the pattern is stochastic in all three spatial dimensions, theat least one patterned surface being created from a pre-patternedsurface on the base by a laser process, wherein the resulting patternedsurface has elevations both above and below the elevation of thepre-patterned surface; applying a metal to the at least one patternedsurface so as to provide at least one metalized patterned surface;positioning the analyte on the at least one metalized patterned surface;and performing a diagnostic assay of the analyte.
 9. A method accordingto claim 8 wherein the nature of the patterned surface is regulated bybeam control.
 10. A method according to claim 8 wherein the nature ofthe patterned surface is regulated by structuring the laser light whichis delivered to the base.
 11. A method according to claim 10 wherein thelaser light is split into two or more beams which are recombined at anangle so as to form a selected pattern on the base.
 12. A methodaccording to claim 10 wherein the laser light is passed through anoptical mask before being delivered to the base.
 13. A method accordingto claim 12 wherein the optical mask is deposited directly on the base.14. A method according to claim 12 wherein the optical mask is spacedfrom the base.
 15. A method according to claim 12 wherein fluid ispassed through the gap which is located between the optical mask and thebase.
 16. A method according to claim 10 wherein a layer ofinhomogeneous material is deposited on the base, and further wherein theinhomogeneous material has inhomogeneities on scales comparable to theresulting structures of the patterned surface.
 17. A method according toclaim 16 wherein the inhomogeneous material is a non-uniform lightabsorber.
 18. A method according to claim 16 wherein the inhomogeneousmaterial is a non-uniform light reflector.
 19. A method according toclaim 8 wherein the analyte comprises a fluid.
 20. A method according toclaim 8 wherein the analyte comprises a solid.
 21. A method according toclaim 8 wherein the base comprises a semiconductor.
 22. A methodaccording to claim 21 wherein the base comprises silicon.
 23. A methodaccording to claim 8 wherein the base comprises a metal.
 24. A methodaccording to claim 8 wherein laser processing is effected using afemtosecond laser.
 25. A method according on claim 8 wherein laserprocessing is effected by delivering laser light to the base at aselected pulse rate, fluence, angle and/or polarization.
 26. A methodaccording to claim 8 wherein the at least one patterned surfacecomprises high-aspect ratio structures.
 27. A method according to claim8 wherein the at least one patterned surface is configured to providelarge electric fields when the analyte is disposed on the at least onemetalized patterned surface and energy is delivered to the analyteand/or the at least one metalized patterned surface.
 28. A methodaccording to claim 8 wherein the metal comprises a metal film.
 29. Amethod according to claim 8 wherein the metal is selected from the groupconsisting of silver and gold.
 30. A method according to claim 8 whereinthe diagnostic assay comprises surface enhanced Raman spectroscopy, andfurther wherein the at least one metalized patterned surface providesthe desired surface enhancement for the analyte.
 31. A method forsensing at least one of the presence and quantity of an analyte, whereinthe method comprises: providing a base comprising at least one patternedsurface, wherein the at least one patterned surface is characterized bystructures ranging in scale from 10 to 2000 nanometers in all threespatial dimensions and further wherein the pattern is stochastic in allthree spatial dimensions, the at least one patterned surface beingcreated from a pre-patterned surface on the base by a laser process,wherein the resulting patterned surface has elevations both above andbelow the elevation of the pre-patterned surface; positioning theanalyte on the at least one patterned surface; and performing adiagnostic assay of the analyte.
 32. A method according to claim 31wherein the at least one patterned surface is subjected to a furtherprocessing step prior to use in performing a diagnostic assay, whereinthe further processing step converts the at least one patterned surfaceinto a substantially pristine layer.
 33. Apparatus for use in performinga diagnostic assay of an analyte, the apparatus comprising: a basecomprising a first surface and a second surface, the first surface beingsubstantially planar and the second surface being characterized bystructures ranging in scale from 10 to 2000 nanometers in all threespatial dimensions, the dimensions of the structures being substantiallystochastic in all three spatial dimensions, the second surface beingcreated from a portion of the first surface by a laser process such thatthe structures have elevations both above and below the plane of thefirst surface; and a metal applied to the at least one patterned surfaceso as to provide at least one metalized patterned surface.
 34. Apparatusfor use in performing a diagnostic assay of an analyte, the apparatuscomprising: a base comprising an unpatterned surface and a patternedsurface, the unpatterned surface being substantially planar and thepatterned surface being characterized by structures ranging in scalefrom 10 to 2000 nanometers in all three spatial dimensions, with thepattern being stochastic in all three spatial dimensions, and with thestructures having elevations both above and below the plane of theunpatterned surface; and a metal applied to the at least one patternedsurface so as to provide at least one metalized patterned surface.